Hemophilia A is an inherited bleeding disorder characterized by the deficiency or dysfunction of Factor VIII (FVIII). FVIII serves as a critical co-factor in the intrinsic pathway of the coagulation cascade. Replacement therapy with recombinant human FVIII (rFVIII) or plasma-derived FVIII is the most common therapy employed in controlling bleeding episodes. However, the induction of neutralizing antibodies against the administered protein in approximately 15-30% of patients is a major complication in therapy [1-3]. The neutralizing antibodies frequently target the C2 domain, which is also involved in binding to phospholipids in vivo.
FVIII is a large multi-domain glycoprotein consisting of domains A1, A2, B, A3, C1 and C2 [4,5]. Systematic epitope mapping studies have revealed that anti-FVIII antibodies mainly target defined regions in the A2 (heavy chain), A3 and C2 domains (light chain) of FVIII [6,7]. The epitope determinant within the A2 domain has been mapped to residues Arg484-Ile-508 [8,9]. Antibodies targeting this region have been shown to inhibit the activated form of FVIII (FVIIIa) by blocking interaction of A2 domain with factor IXa (FIXa) [10]. The major epitope determinant within the A3 domain comprises residues 1811-1818 and antibodies against this region also prevent interaction of FVIII with FIXa resulting in loss of cofactor activity [11]. The epitope determinants within the C2 domain have been mapped to residues 2181-2312 [12,13] which encompass the immunodominant, universal CD4+ epitopes, 2191-2210, 2241-2290, 2291-2330 [14,15]. Antibodies against the C2 domain interfere with the binding of FVIII to platelet membrane surface, which is rich in phosphatidylserine (PS), that is essential for the amplification of the coagulation cascade.
Because of the immune response, generated against administered Factor VIII, there is a need for identification of formulations in which the immunogenicity of Factor VIII is reduced preferably without adversely affecting the circulating half life.